Ribonucleoproteins, or RNA/protein complexes (RNPs), are associated with several human diseases including autoimmune disorders such as lupus and arthritis. RNPs are essential components of all cells. They play a critical role in many cellular functions, but perhaps the most important is in protein synthesis. RNPs are involved in transcription; i.e., they are the primary component of the ribosome, where protein translation occurs, and they are responsible for producing of mature transfer RNA (tRNA) molecules. This last essential function is performed by the enzyme ribonuclease P (RNase P), which removes the 5' leader of the premature RNA molecule to produce a mature tRNA. Bacterial RNase P consists of one 300-400 nucleotide RNA subunit and one -120 residue protein molecule. Very little is known about the cellular mechanisms for the assembly of RNPs in general and RNaseP specifically. The goal of the proposed research is to elucidate the kinetic and thermodynamic mechanisms of bacterial RNaseP assembly under physiological conditions. Dr. Oas' preliminary studies have shown that the protein subunit is denatured in the absence of an anionic ligand. Dr. Oas hypothesizes that RNase P protein has evolved to be natively unfolded, in order to enhance its ability to assemble into the holoenzyme complex, either kinetically or by increasing the specificity of its interaction with RNaseP RNA. Dr. Oas will test this hypothesis by comparing small molecule and oligonucleotide binding. Dr. Oas will also determine the relative energies of three states of the protein: denatured, unliganded native and liganded native. Dr. Oas will also measure the relative rates of folding and binding and test his hypothesis that ligand binding occurs after folding. In addition, he will compare the structures of unliganded native protein to that of protein bound to small molecule ligands, olgonucleotides and RNase P RNA. This in vitro work will make possible future experiments on the assembly of RNaseP and other RNPs in the cell.